补阳还五汤对大鼠局灶性脑缺血损伤的长期保护作用

Long-term cerebroprotective effects of buyang huanwu decoction in rats after focal cerebral ischemia injury

目的:观察补阳还五汤对大鼠脑缺血损伤后感觉运动功能和脑损伤的长期影响。方法:实验于2005-06/08在浙江中医学院实验动物中心完成。①取33只健康雄性SD大鼠,随机分为3组:补阳还五汤组(n=12)、缺血组(n=11)和假手术组(n=10)。前2组采用线栓法诱导大鼠大脑中动脉阻塞模型;假手术组只分离血管,不插栓线。补阳还五汤组:从缺血前7天开始按13.3g/kg剂量灌服补阳还五汤水煎剂(补阳还五汤处方来源于《医林改错》,成分:黄芪120g,当归6g,川芎4.5g,赤芍4.5g,地龙3g,红花3g,桃仁3g,药材购自浙江中医学院附属门诊部并经鉴定,煎2次,40min/次,合并后浓缩为含生药2g/mL),持续到缺血后14d,1次/d;其余各组同时灌等量生理盐水,干预时间同补阳还五汤组。②通过肢体放置实验[包括7个步骤。评分标准:正常,2分;延迟(2s)和/或不完全,1分;不反应,0分]在缺血后1～7,10,14,21和28d评价前后肢对触觉和本体感觉的反应。通过横木行走实验(用噪音刺激大鼠跨过横木。评分标准:大鼠不能呆在横木上,0分;大鼠能呆在横木上但不动,1分;大鼠试图跨过横木但摔下,2分;大鼠跨过横木,但损伤的后肢滑落次数超过50%,3分;超过1次但不到50%,4分;仅滑落1次,5分;顺利通过,6分)评价运动的协调和整合缺损。通过黏胶揭除实验(评分标准:1分为≤10s;2分为10～19s;3分为20～29s;4分为30～39s;5分为40～49s;6分为50～59s;7分为≥60s)评价躯体感觉功能。2块同面积的医用胶布分别贴在两前肢腕部腹侧面作为触觉刺激,记录大鼠揭除胶布的潜伏期。③缺血后第29天处死大鼠,取脑,冠状冰冻切片,采用甲苯胺蓝染色测定梗死体积。④肢体放置和横木行走实验结果差异比较采用非参数Mann-WhitneyU检验,黏胶揭除实验结果和梗死体积差异比较采用单因素方差分析。结果:大鼠33只均进入结果分析。①肢体放置实验评分:缺血后1～10,14,21,28d缺血组明显低于假手术组(u=0～9,P<0.01)。补阳还五汤组大鼠在缺血后1～7,10,14d明显高于缺血组(u=4.5～35,P<0.05～0.01)。②横木行走实验评分:缺血后1～7d缺血组明显低于假手术组(u=0～10,P<0.05～0.01)。缺血后1～3d补阳还五汤组明显高于缺血组(u=26.5～31,P<0.05)。③黏胶揭除实验评分:缺血后1,7,14,21,28d缺血组明显高于假手术组(t=2.56～21.91,P<0.05～0.01)。缺血后1,7,14,21d补阳还五汤组明显低于缺血组(t=2.36～3.88,P<0.05～0.01)。④脑梗死体积:缺血后29d,补阳还五汤组明显小于缺血组[(95±25),(123±22)mm3,t=2.75,P<0.05]。结论:补阳还五汤能显著改善脑缺血大鼠长期感觉运动功能,减轻脑损伤。

AIM： To observe the long-term cerebroprotective effects of Buyang Huanwu decoction （BYHWD） on the sensorimotor function and brain damage after focal cerebral ischemia in rats. METHODS： The experiment was carried out in the Experimental Animal Center of Zhejiang College of Traditional Chinese Medicine between June and August 2005.①Totally 33 healthy male SD rats were randomly divided into 3 groups： BYHWD group （n=12）, ischemia group （n=11） and shamoperated group （n=10）. The rat models of middle cerebral artery occlusion （MCAO） in the former two groups were induced by the filament method while only blood vessels were detached without filament in the shamoperated group. The administration of BYHWD （13.3 g/kg, consisted of 120 g astragalus root, 6 g Chinese angelica, 4.5 g szechwan lovge rhizome, 4.5 g red peony root, 3 g earth-worm, 3 g safflower and 3 g peach seed, all the herbs were supported and evaluated by the Outpatient Department Affiliated to Zhejiang College of Traditional Chinese Medicine and fried once for 40 minutes, totally for twice and concentrated into 2 g/mL crude drug, with the prescription from Yilin Gaicuo） was taken orally in the BYHWD group while the saline of same dose were given in other groups, once a day from day 7 before ischemia to day 14 after ischemia. ② The limb-placing test （including 7 procedures, score standard： 2 as normal; 1 as delayed for 2 seconds and/or incomplete;0 as no response） was used to evaluate the response of forelimb and hindlimb to the taction and proprioception at 1-7, 10, 14, 21 and 28 days after ischemia. The motor coordination and integrated defect were detected by crussrail-walking test （the rats were stimulated by the noise to pass the crossrail, score standard： 0 as not staying on the crossrail; 1 as staying on the crossrail without moving; 2 as trying to pass but felled： 3 as passing the crossrail but the number of injured hindlimb slipping times 〉 50%; 4 as slipping more than once but 〈 50%; 5 as slipping only once; 6 as passing successfully）. The adhesive tape exposed test （score standard： 1 as ≤10 seconds;2 as 10-19 seconds; 3 as 20-29 seconds; 4 as 30-39 seconds;5 as 40-49 seconds; 6 as 50-59 seconds;7 as ≥ 60 seconds）was performed to measure the somesthesia function. Two medical tapes of same volume were pasted to the ventral side of arm in forelimb respectively and taken as the tactile stimulation to record the latent period of exposing tapes. ③At day 29 after ischemia, the rats were killed to take out the brain and make coronal freezing section. Total infarcted volumes were detected by toluidine blue staining.④The difference between the limb-place test and crossrail-walking test was compared by Nonparametric Mann-Whitney U test while the difference between the adhesive tape exposed test and infarcted area was compared by the oneway analysis of variance. RESULTS： Totally 33 rats were involved in the analysis. ①The score of limb-placing test： It was significantly lower in the ischemia group than in sham-operated group at days 1-10,14,21 and 28 after ischemia （u=0-9,P 〈 0.01）. The score was higher in the BYHWD group than in the control group at days 1-7, 10 and 14 after ischemia （u=4.5-35,P 〈 0.05-0.01）. ② The score of crossrail-walking test： The score was significantly lower in the ischemia group than in the sham-operated group at 1-7 days after ischemia （u=0-10,P 〈 0.05-0.01）. Compared with the ischemia group, the performance of BYHWD treated rats significantly improved at 1-3 days after ischemia （u=26.5-31,P 〈 0.05）. ③The score of adhesive tape exposed test： At 1,7,14,21 and 28 days after ischemia, the score in the ischemia group was higher than that in sham-operated group （t =2.56-21.91,P 〈 0.05-0.01）. Compared with the ischemia group, the score of BYHWD treated rats was significantly lowered at 1,7,14 and 21 days after ischemia （t=2.36-3.88,P 〈 0.05-0.01）. ④ Infarcted volume： Brain infarcted volume was significantly lower in the BYHWD group than in the control group at 29 days after ischemia [（95±25）, （123±22） mm^3, t=2.75,P 〈 0,05]. CONCLUSION： The BYHWD can remarkably improve the sensorimotor function and relieve brain damage after local ischemia.